Little attention has been paid to customising fatigue questionnaires for patients with Substance Use Disorders (SUDs). The present study aims to validate and shorten the nine-item Fatigue Severity Scale (FSS-9) and Visual Analogue Fatigue Scale (VAFS) for use with this population.We used data from a nested cohort with annual health assessments with responses on the FSS-9 and VAFS. During the period 2016–2020, 917 health assessments were collected from 655 patients with SUD in Bergen and Stavanger, Norway. A total of 225 patients answered the health assessment at least twice. We defined baseline as the first annual health assessment when the health assessments were sorted chronologically per patient. We checked for internal consistency, and we used longitudinal confirmatory factor analysis (CFA) and linear mixed model (LMM) analysis to validate and shorten the FSS-9 and VAFS.2 = 13.33, degree of freedom = 8, P = 0.101). The LMM analysis showed equal linear changes at the individual level for the FSS-3  and FSS-9  between the health assessments.The internal consistency of the FSS-9 was excellent with a Cronbach’s α of 0.94 at baseline and 0.93 at the second annual health assessment. When shortening the FSS-9 to a three-item FSS , the Cronbach’s α was 0.87 at baseline and 0.84 at the second health assessment. The internal consistency was not affected when the VAFS was added to the FSS-3 and the FSS-9. The longitudinal CFA model showed a well-fitting model for the FSS-3 (χThe FSS-9 could be shortened to the FSS-3 with high validity and reliability for patients with SUDs and the addition of VAFS did not provide much added variability. Fatigue is a common subjective health complaint that significantly affects patients with chronic diseases –3. For pPatients with SUDs often live in a chaotic situation with extensive medical and psychosocial health challenges, including polysubstance use, substance intoxications and withdrawals, psychiatric comorbidities , chronic viral hepatitis, financial risk, and temporary living situations , 8. ThisThe nine-item Fatigue Severity Scale (FSS-9) and the Visual Analog Fatigue Scale (VAFS) are two well-known fatigue questionnaires. The evidence for the validity of the FSS-9 has been obtained for a wide range of chronic infectious and neurological diseases—such as hepatitis C virus infection , ParkinsWith this validation study we sought to address two gaps in the literature on the assessment of fatigue in patients with SUDs: (1) to evaluate whether the scores produced by a shortened version of FSS-9 can be interpreted similarly to the scores produced by the FSS-9, and (2) to determine whether there is value to adding the VAFS to either the FSS-9 or the shortened version of FSS-9.We hypothesised that the FSS-9 and VAFS questionnaires can create a short version of the FSS-9 which demonstrates strong evidence of reliability and validity when used with patients who have SUDs.We drew data from a nested cohort from the INTRO-HCV trial that collected data on patients with SUDs in Bergen and Stavanger, Norway . We recr®) under the supervision of research nurses.All included patients were invited to an annual health assessment, including FSS-9 and VAFS measurements and a survey of their current sociodemographic situation. We collected all data in a health register using data collection software : 4 months)  to 7 (worst fatigue) and the VAFS was answered by placing a mark on a line from 0 (no fatigue) to 10 (worst fatigue) that represent the fatigue level. The data collection software only allowed valid responses for each question and prompted for responses to unanswered questions before submission in order to minimise missing data. In a previous study, the US-English version of the FSS-9 has been translated into Norwegian by a qualified native Norwegian-speaking translator and back-translated into US-English by an authorised native US-English-speaking translator , for confirmatory factor analysis (CFA), and for linear mixed model (LMM) analysis . The threshold for statistical significance was set to P < 0.05 for all analyses unless otherwise stated.We used Stata/SE 16.0  for descriptive analysis and IBM SPSS version 24.0 and MWe calculated the internal consistency of the FSS-9 and this scale including the VAFS at baseline and at the second health assessment. As a part of the validation study, we explored whether there was value in adding the VAFS to the FSS-9 by evaluating if the VAFS added more information than captured by the FSS-9. Cronbach’s α was considered to show good internal consistency if Cronbach’s α was above 0.70 –24. We t2, degrees of freedom, p values, Comparative fit index, Tucker Lewis Index, Root Mean Square Error of Approximation with 90% confidence interval, and the probability of close fit. A well-fitted model should have a statistically non-significant χ2, values of Comparative fit index and Tucker Lewis Index should be above 0.95, and Root Mean Square Error of Approximation should preferably be below 0.05 (close fit) [2 difference test). We analysed all variables as continuous variables due to the relatively high number of categories in the ordinal variables . The CFAs were run using the Robust Maximum Likelihood estimator. According to previous studies showing good reliability and strong inter-item correlations of the FSS-9 and this scale including the VAFS [We used CFA models to test the structure of the items in the FSS-3 and FSS-9, and these scales including the VAFS at baseline and at the second health assessment in order to evaluate the relationships between the items and their underlying latent factors , 25–28. ose fit) . Root Meose fit) . We usedthe VAFS , 16, we the VAFS . Measureplus multilevel modelling: TwoLevel) to evaluate linear changes from baseline in the sum scores of the FSS-3 and FSS-9, the scores in the separate FSS-9 items, and the VAFS score. We included all 917 health assessments. First, we estimated a full random intercept random slope model, which gave us the mean and individual variance in terms of both level and change together with the relationship between level and change [plus Maximum Likelihood Robust estimator to correct standard errors for potential deviation from normality [We used a LMM analysis  West, Norway . Each patient provided written informed consent prior to enrolling in the study.Seventy-one percent were males, and the mean age was 43 years Table . Half haThe nine-item Fatigue Severity Scale’s Cronbach’s α was 0.94 at baseline and 0.93 at the second health assessment  = 1, P < 0.001). The factor loadings for the nine items of the FSS-9 in this model were (ranged from items 1 to 9): 0.66, 0.74, 0.77, 0.78, 0.85, 0.79, 0.84, 0.84, and 0.84. The FSS-3 showed a well-fitted model. The FSS-3 and FSS-9 were highly correlated with r = 0.95, P < 0.001, giving 90% explained variance after estimating factor scores. The VAFS, together with the FSS-3 and FSS-9, respectively, gave identical results. The correlations with VAFS were r = 0.68, P < 0.001 (FSS-3) and r = 0.70, P < 0.001 (FSS-9). We obtained relatively similar results for the factor models at the second health assessment for both the FSS-3 and FSS-9 with and without the VAFS. The full FSS-9 version showed the factor loadings to be (ranged from items 1 to 9) 0.59, 0.66, 0.75, 0.69, 0.84, 0.74, 0.85, 0.80, and 0.79. The longitudinal CFA model based on the FSS-3 supported time-invariant equal factor loadings and equal residuals between the baseline and the second health assessment  to three (FSS-3). Cronbach’s α is related to the ratio between the mean covariance and the total variance, and the number of items, which makes the use of α thresholds to some degree arbitrary . Our resThe confirmatory factor analyses demonstrated unidimensional models for the FSS-9 at baseline and at the second health assessment, which was improved when adding the residual covariance between items 2 and 3. This means that the single-factor model did not fully capture these items’ responses. The explanations for this might be related to similar phrasing and wording , as wellThe linear mixed model analysis showed that items 5–7 included in the FSS-3 remain substantially stable and equal over time between the annual health assessments compared to the separate items 1–4 and VAFS. The result might give further arguments for the better validity of the FSS-3 questionnaire, and the FSS-3 is assumed to be less sensitive to fluctuations compared to the FSS-9. This points out that the FSS-3 might be preferred when evaluating changes in fatigue over time in the SUD population.This study had some strengths. First, we collected data from patients who are difficult to reach in both research and health care. Of those, 225 patients were followed up by two or three annual health assessments, making longitudinal analyses possible. Second, patients recruited to this study answered the FSS-9 and VAFS questionnaires under different mental and psychosocial conditions, for example, when they were going through substance intoxications or withdrawal and living on the street, which might increase the generalisation of the results. The study also had some limitations. First, the majority of the patients were recruited from opioid agonist therapy, making this validation study more transferable to opioid agonist therapy populations than other substance dependence populations. Second, the FSS-9 questionnaire used was translated from US-English into Norwegian and back-translated into US-English by native Norwegian and US-English-speaking translators , but as The present study demonstrates that the FSS-9 can be shortened to just the FSS-3 among patients with SUDs. The value in adding the VAFS to the FSS-3 and FSS-9 did not provide much added variability. We found that the FSS-3 was more consistent in the structure of changes in fatigue levels compared to the FSS-9. The FSS-3 seems to be a useful patient-reported outcome measure of fatigue in this population at a group level, although the clinical relevance at an individual level remains more controversial.Additional file 1. The number of months from baseline to the second or third measurements. No.: Number of patients; SD: Standard deviation; ref.: Reference. Tables display the number of patients with one, two, and three health assessments, including the Fatigue Severity Scale and the Visual Analog Fatigue Scale measurements. The table displays the time interval between the second and third health assessments and baseline.Additional file 2. The US-English and the Norwegian versions of the nine-item Fatigue Severity Scale. FSS-9: Nine-item Fatigue Severity Scale. All items in the FSS-9 are answered with a Likert scale from 1 to 7 where 1 indicates “strongly disagree” and 7 indicates “strongly agree”.Additional file 3. The Cronbach’s α if-item-deleted and Item-Total correlation for the FSS-3 and FSS-9, and these scales including the VAFS. FSS-9: Nine-item Fatigue Severity Scale; FSS-3: Three-item Fatigue Severity Scale; VAFS: Visual Analogue Fatigue Scale; No.: Number of patients. *Due to high internal consistency and almost equal Cronbach’s α of the remaining items, we deleted item 8 using clinical judgement. Item 8 could be difficult to answer if patients were affected by substances or were going through substance withdrawals. **We deleted item 9 using clinical judgement due to high internal consistency and almost equal Cronbach’s α in the remaining items and the fact that unemployment was frequently reported among patients with SUDs. Tables a) and c) show the Cronbach’s α if-item-deleted for the FSS-3 and FSS-9, and these scales including the VAFS using data from the first health assessment (baseline) (Table a) and the second health assessment (Table c). Tables b) and d) show the Item-Total correlation for the FSS-3 and FSS-9, and these scales including the VAFS using data from the first health assessment (baseline) (Table b) and the second health assessment (Table d). The values in bold represent the FSS-3’s Cronbach’s α if-item-deleted and Item-Total correlation.


After 20-pulsed irradiation, the results show that only Ni3Nb clusters with around 3 nm in size were dispersed in fully amorphization layer. With increased pulse number to 30, the nano-particles embedded into the amorphous layer were grown up, the size of which was about 8 nm. The microstructure evolution during HCPEB irradiation was from the partial amorphous to fully amorphous and then to nano-crystallization. The 20-pulsed samples possessed the best hardness and corrosion resistance. The ultrafine clusters uniformly embedded into amorphous layer were main reason for improving properties.In this paper, the Ni-Nb coatings were successfully prepared onto the GH3039 alloys by High current pulsed electron beam (HCPEB). The transmission electron microscopy (TEM) results confirmed that the Ni-Nb layer of 10-pulsed samples exhibited partial amorphization, which was consisted of γ-Ni particles, rod-like Ni Amorphous structures have been prepared in the binary Ni-X systems, while Ni-Nb system is one of the best binary metallic glass forming systems due to their unique properties including excellent properties, high corrosion resistance and various magnetic properties ,2,3. Ni-The above problems could be solved by the formation of the alloying layer with amorphous structures in nanoscale size on the substrate. The alloying layer could be achieved by High current pulsed electron beam (HCPEB) irradiation as an innovative processing technique, which has been developed in recent years. Generally, this technique with many characteristics, such as short-duration pulse, high energy etc., was adopted to complete surface treatment. As inputting extremely intensive energy onto the topmost surface, the material surface was melted quickly and then solidified owing to the heat extraction towards the sub-surface. Under the effect of ultrafast melting and solidification, refined grains, even nano-grains and amorphous phase were formed which could be realized to promote the improvement of material properties ,18,19. HTherefore, in present research, the new Ni-Nb amorphous alloys have been prepared onto the GH3039 alloys by HCPEB irradiation. The aim of the present work was to investigate the microstructure evolution of Ni-Nb alloying layer for different number of pulses and their corresponding hardness and corrosion property.3 in bulk-shaped were selected as substrate. The GH3039 superalloy was composed of C ≤ 0.08%, Mn = 0.40%, Mo = 1.80%, P = 0.02%, Al/Ti = 0.75%, Fe = 3.00%, Si = 0.80%, S = 0.012%, Cr = 22.00%, Nb = 1.30%, and Ni = balance. Before preparation of coating, all the samples were mechanically grounded with SiC papers (with 5.5 μm particle size), and then polished (with 1 μm diamond paste), finally cleaned. A mixture with chemical composition Ni62Nb38 was made by mixing commercially Ni (99.99% in purity) and Nb (98.0% in purity) powder. The mixed powder, GH3039 sample and a number of stainless-steel grinding balls were filled into the grinding vial. The prepared process was grinding duration of 3 h within argon gas at room temperature. As-fabricated single Ni-Nb coating was obtained onto the topmost of GH3039 alloy. After that, the as-fabricated coating was irradiated at room temperature using a HOPE-I type HCPEB device. The corresponding HCPEB parameters were the electron energy 27 keV, the beam diameter 60 mm, and the vacuum 5.0 × 10−3 Pa, energy density of 6 J/cm2, pulse duration of 1.5 μs and interval between each pulse of 10 s. The fabricated and treated process was schematically exhibited in Ni-based GH3039 superalloy alloys with 10 × 10 × 5 mm2 in area was tested with a CHI760C electrochemical workstation  in the 3.5 wt% NaCl (0.6 M) water solution. The cyclic polarization was done at a sweep rate of 0.333 mV/s. A saturated calomel electrode, platinum and Ni-Nb coating were served as the reference electrode, the counter electrode and the work electrode, respectively.The X-ray diffraction  was carried out on the CuKa radiation in a D/max-2500/pc X-ray diffractometer to verify phase structures. The surface morphology of Ni-Nb coating was observed by using FEI Nova Nano 450 scanning electron microscope  with an Inca energy 350 energy-dispersive spectrometer (EDS) at 15 kV acceleration voltage. The microstructure was carried out on the JEM-2100 transmission electron microscopy  in the combination with selected area electron diffraction (SAED). The mechanical property of Ni-Nb coating was examined by an HVS-1000 microhardness measurement device  with a load of 0.245 N (25 g) applied for 15 s. The corrosion property of the Ni-Nb coating with 1 cm3Nb phase in the irradiated layer. Noticeably, Nb peak at 2ϴ = 38° with low intensity still existed in the XRD pattern after 10-pulsed irradiation. With pulses increased to 20, the Nb peak was disappeared, which suggests the formation of Ni3Nb or Ni(Nb) solid solution. Additionally, Ni diffraction peaks have shifted to low angle after 20-pulsed irradiation comparing to the initial sample, meaning that the Ni lattices were expanded, which indicates that Ni(Nb) solid solution was formed. For the 30-pulsed samples, a minor shift of Ni peaks to high angle occurred. The possible reason was that the Nb atoms were decomposed from the Ni(Nb) solid solution to form Ni3Nb phase. Especially in the pattern of 30-pulsed Ni-Nb samples, the δ-Ni3Nb phase was clearly identified. Besides, the broader diffractograms peaks of Ni were resulted from the formation of amorphous phase due to the distorted lattice. Certainly, all of the grain refining, dislocation density increasing and micro stress accumulating resulted into broader peaks. The specific reason needs further detailed characterization by TEM to elaborate in the following.3Nb phase  and the Fast-flourier transformation (FFT) pattern of enlarged image was exhibited in the 3Nb, and for the B zone, d(111) = 0.203 nm and d(200) = 0.176 nm for the FCC Ni phase. Therefore, it could be concluded that nano black particles were the mixture of γ−Ni particles and Ni3Nb particles with the blocky-shaped structures.In addition, a large number of nano black particles and blocky-shaped structures with about 30 nm in size were formed in some area, as shown in Nb phase d. For fu3Nb clusters. 3Nb particles. In addition, different from the 10-pulsed samples, no large size Nb particles were observed in the 20-pulsed layer, which suggests that Nb was completely dissolved into the Ni amorphous matrix.3Nb. Noticeably, faint diffraction speckles were observed in 3Nb and Nb particles, as shown in 3Nb phase. Therefore, it can be concluded that the alloying layer was consisted of nano Νb and Ni3Nb particles that were uniformly dispersed into the Ni-Nb amorphous matrix after 30-pulsed irradiation.After 30-pulsed irradiations, a little change in microstructure took place in the alloying layer. There was also consisted of amorphous matrix and the nano particles embedded in it, but the size of which increased to about ~8 nm, as shown in 60Nb40 coatings due to HCPEB irradiation. However, from 3Nb with large size dispersed in the amorphous Ni-Nb layer  > Ni (0.125 nm), together with the highly negative mixing enthalpy in the Ni-Nb (−30 kJ/mol) [3Nb clusters have grown to only ~3 nm in size, as shown in Based on the experimental results and observations at different stages of HCPEB irradiation, a microstructure evolution is proposed for HCPEB irradiation, as shown by a schematic in Nb layer b. Hence,ayer see . Therefoayer see c. The mo kJ/mol) ,27. All 3Nb clusters coarsened up to 8 nm in size with increasing pulses number. Meanwhile, a large number of Nb nano-particles has already been formed during this stage. This strongly supported that the chemical fluctuation to break the Ni/Nb component ratio (Ni:Nb = 62:38) in the local area. It is considered that the growth of Ni3Nb particles is due to composition fluctuation existed in the liquid metal [3Nb nano-particles, as shown in 62Nb38 alloying layer also to break the Ni/Nb component ratio (the best forming amorphous ratio of Ni to Nb is 62:38), which makes it to deviate proper component of amorphous phase. Finally, the formation of nano-crystalline inevitably took place to decrease the content of amorphous phase for 30-pulsed Ni-Nb layer. Therefore, it could be concluded that the microstructure evolution proposed here was partial amorphous to fully amorphous and then to nano-crystalline during HCPEB irradiation.On base of the stage II, the size of nano-Niid metal . Owing t3Nb particles resulted in the increasing strengthen, enabling the Ni-Nb layer hardening. It was understandable that the decreasing hardness of 30-pulsed samples was owing to the precipitation of Nb nano-particles, damaging reduction of amorphous phase strengthen. Meanwhile, the coarsening of dispersed Ni3Nb nanocrystals occurred during HCPEB irradiation. So, these factors were making hardness for the 30-pulsed samples to decrease. In conclusion, samples hardening was mainly attributed to the metal-metal interatomic bonding strengthen, nanocrystalline strengthen and so on [To investigate the mechanical property of Ni-Nb coating, the microhardness testing was performed. nd so on ,30.corr) and corrosion potential (Ecorr) were calculated and tabulated in corr and the Icorr of the initial samples were −0.990 V and 28.90 μA·cm−2, respectively, as shown in corr and Icorr of specimens were changed apparently. The Ecorr of 10- and 20-pulsed samples was increased to −0.904 V and −0.799 V and Icorr decreased to 10.2 μA/cm2 and 6.98 μA/cm2, respectively. As increasing the pulses number up to 30, the Icorr increased to 7.83 μA/cm2 and the Ecorr decreased to −0.887 V. Therefore, a drastic enhancement of corrosion resistance was obtained for the Ni-Nb layer with 20 pulses compared to that of other samples.Generally, the corrosion resistance of amorphous alloys was superior to the crystalline metal material. However, the Ni-Nb alloys only with amorphous structure have a characterization of the cracks in the reference . It destThe main findings are summarized as follows:1. The dense Ni-Nb coating with ~1.9 μm in thickness was fabricated onto GH3039 alloys, in which the distribution of Ni and Nb elements was not uniform. After HCPEB irradiation, the amorphous layer was fabricated onto the substrate, the thickness of which was increased with increasing number of pulses. The layer could be divided into re-melted layer and heat affected layer that was about 2.5 μm in thickness for the 30-pulsed sample.3Nb particles with 30 nm in size existed in the alloying layer. There were also δ−Ni3Nb particles characterized by rod-like structure distributed in the amorphous matrix.2. After 10-pulsed irradiation, the whiter Nb particles and ultrafine black particles corresponding to γ-Ni particles and Ni3Nb particles in large size were dissolved to form Ni3Nb in nanometer uniformly distributed into the amorphous layer. Compared to the 20-pulsed sample, the size of nano-particles embedded into the amorphous layer was increased after 30-pulsed irradiation, reached to about ~8 nm.3. In 20-pulsed samples, Nb particles and Ni4. SAED patterns of the Ni-Nb layer changed from three broader rings with low diffusion to only two diffraction rings and then to amorphous halo coexisted with several weak diffraction speckles. Combined with the microstructure, the structure evolution during HCPEB irradiation could be described as from the partial amorphous to fully amorphous and then to nano-crystallization.3Nb particles in nano size uniformly distributed in the amorphous layer.5. The hardness and corrosion resistance of Ni-Nb amorphous layer were significantly improved after HCPEB irradiation. The 20-pulsed sample obtained the best results. The main factor contributing to such improvement was the formation of Ni


Due to improved survival upon effective anti-cancer therapies, the management of treatment-related side-effects is of increasing interest and importance. Cardiovascular side-effects of chemo-, targeted- and/or immunotherapies are common and can be harmful. To date, the identification of patients who could experience those cardiovascular side-effects prior to the anti-cancer therapy start is difficult. We show that the use of a simple electrocardiographic (ECG) score can help to predict the occurrence of cardiovascular toxicity of anti-cancer therapies.n = 25), 19% reached 0 points, 50% (n = 67) reached 1 point, 25% (n = 33) reached 2 points, 5% (n = 7) reached 3 points and 0.7% reached 4 or 5 points (n = 1 respectively). The incidence of cardiotoxicity (n = 28 [21%]) increased with the ECG score, with 0 points at 0%, 1 point 7.5%, 2 points 55%, 3 points 71% and ≥3 points 50%. In the ROC (Receiver operating curves) analysis, the best cut-off for predicting cardiotoxicity was an ECG score of ≥2 points  which was then defined as a high-risk score. High-risk patients did not differ concerning their age, LV ejection fraction, classical cardiovascular risk factors or cardiac biomarkers compared to those with a low-risk ECG score. Conclusion: ECG scoring prior to the start of anti-cancer therapies may help to identify therapy-naïve cancer patients at a higher risk for the development of cardiotoxicity.Objective: To evaluate a new electrocardiographic (ECG) score reflecting domains of electrical and structural alterations in therapy-naïve cancer patients to assess their risk of cardiotoxicity. Methods: We performed a retrospective analysis of 134 therapy-naïve consecutive cancer patients in our two university hospitals concerning four ECG score parameters: Contiguous Q-waves, markers of left ventricular (LV) hypertrophy, QRS duration and JTc prolongation. Cardiotoxicity was assessed after a short-term follow-up (up to 12 months). Results: Of all the patients ( Risk prediction for cardiotoxicity in cancer patients is an important aspect in the field of cardio-oncology. Several parameters have previously been associated with an increased risk for cardiovascular adverse events from cancer therapy including age, sex, pre-existing cardiac diseases  dysfunction and coronary artery disease (CAD)), arterial hypertension, specific chemotherapy drugs and regimens, their cumulative dose and previous or concomitant chemo- and/or radiation therapies ,2,3,4,5.This retrospective investigation included 134 consecutive therapy-naïve cancer patients who were examined in our two cardio-oncology units  between 2018 and 2019 before the start of chemo- and/or immunotherapy  decreased by 15% according to current recommendations .Twelve-lead electrocardiograms (ECGs) were recorded using GE Healthcare ECG machines . Assessment of the ECG score was done in accordance with the original publication in which the score was developed  15]. In. In15]. p-value < 0.05 was considered statistically significant.Continuous variables are shown as the mean ± standard deviation (SD). Normal distribution was tested by the D’Agostino & Pearson omnibus normality test. Two independent groups were compared using the student’s t-test for normally distributed variables and using the Mann–Whitney U test for nonparametric parameters. Receiver operating curves (ROC) were determined to analyze the predictive potential of the ECG score concerning cardiotoxicity, and the area under the curve (AUC) was calculated. The Youden index was calculated to identify the ECG score cut-off value for cardiotoxicity and proportions were compared using the Chi-square test. Correlations between parameters were analyzed by Spearman rank correlation and multiple testing was corrected by the Bonferroni correction. Sensitivity analysis was performed using Fisher’s exact test. Data were analyzed using Prism software . A n = 18).The baseline characteristics of the study population are provided in The ECG score was calculated in all 134 patients  and 25 pContiguous Q waves were present in 4% of the patients, 18% showed a QRS duration of ≤80 ms, 69% 80–110 ms and 13% had a QRS duration > 110 ms. ECG signs of LV hypertrophy showed in 13% of the patients and a JTc duration of >360 ms was seen in 28% of the study patients .p < 0.0001, n = 92/42, p < 0.0001).The incidence of cardiotoxicity in the whole study cohort was 21%. The incidence of cardiotoxicity during the follow-up was 0% in patients with 0 points in the ECG score (0/25), 7.5% in patients with 1 point (5/67), 55% in patients with 2 points (18/33), 71% in patients with 3 points (5/7), 100% in the 4-points patient (1/1) and 0% in the patient with 5 points (0/1) A. The sen = 92/42, p < 0.05, n = 42/92. p < 0.01, n = 42/92, p < 0.01, n = 92/42, p = ns, The two risk groups did not differ concerning age, sex, LV-EF, or laboratory values, e.g., hemoglobin, platelet count, creatinine, hsTnI and NT-proBNP . C-reactp = 0.0435, The Spearman rank correlation showed that the ECG score was not associated with age, sex, BMI, heart rate, presence of classical cardiovascular risk factors, intake of betablockers or ACE-I/ARB, hsTnI and NT-proBNP or LV-EF . There wWe have presented the descriptive data on a simple ECG score by Chatterjee et al. in therapy naïve-cancer patients . We showThe original score was developed to predict the incidence of SAD in CAD patients, but the single parameters of this ECG score represent structural and electrical alterations which are not specific for ischemic heart disease and could therefore be translated to several cardiac abnormalities or potentially cardiotoxic interventions. Some of these parameters were already validated in heart failure patients without ischemic heart disease , while oThis study is the first to evaluate an ECG-based score concerning therapy-naïve cancer patients and its potential to predict cardiotoxicity. High ECG scores were associated with high incidences of cardiotoxicity. Higher patient numbers would be necessary to validate these results for the group with four or five points. Although the score was designed and validated for the prediction of mortality in CAD patients, contingency analyses and receiver operating curves showed high sensitivity, specificity and accuracy to predict cardiotoxicity in the present study . While tIn this study, 21% of the patients had cardiotoxic side-effects of their anti-cancer therapies. This rate seems high but can be explained by two reasons. First, many patients were treated with anthracyclines, many of them in combination with alkylating agents and furthermore showed other factors of higher rates of cardiotoxicity such as female sex, preexisting arterial hypertension and higher ages . We inclThe ECG score was not associated with markers of cardiac damage or volume overload like hsTnI and NT-proBNP before the start of anti-tumor therapies. This could be due to the fact that most of the patients had normal biomarker levels at presentation. The mean hsTnI value was 6 ng/L with only 3% of the patients having an hsTnI level above the upper limit of normal . The rolThe present study has several limitations. The first is the fact that 13–18% of the patients showed classical cardiovascular risk factors, e.g., diabetes or smoking, while half of the patients suffered from preexisting arterial hypertension and were being treated accordingly . This man = 134 were included, the results show good values for sensitivity and AUC.Although the study results are further limited by the retrospective design and although relatively low patient numbers of only We have reported that around one-third of typical therapy-naïve cancer patients presenting in cardio-oncology are at a high risk of developing cardiotoxicity according to a newly developed ECG score. The evaluation and consideration of this new ECG score in the setting of cardio-oncology could be helpful to identify patients at a higher risk of developing cardiotoxic side effects. Although our data is hypothesis-generating, we think that the assessment of the ECG score may be a new component of personalized precision medicine in cardio-oncology. Accurate tools for the prediction of cardiotoxicity could, e.g., shorten the follow-up routines for high-risk patients or lead to an early start of a preventive drug therapy in those patients. Prospective long-term data are needed to confirm reliable factors for the prediction of cardiotoxicity in cancer patients.


Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments. Plant mitochondrial genomes have very low base substitution rates but expand and rearrange rapidly ,2,3,4,5.Much less is known about the multiple pathways of DNA repair in plant mitochondria than in other systems, such as the nucleus. So far, there is no evidence of nucleotide excision repair (NER) or mismatch repair (MMR) in plant mitochondria ,13. It hArabidopsis thaliana UNG knockout line and investigate the effects on the mitochondrial genome over many generations. To disrupt the genome further, we express the cytidine deaminase APOBEC3G in the Arabidopsis mitochondria (MTP-A3G) to increase the rate of cytosine deamination and to accelerate DNA damage.In light of the apparent absences of NER and MMR in plant mitochondria, it is possible that many lesions, including mismatches, are repaired by creating double-strand breaks and by using a template to repair both strands. Our hypothesis is that DSBR accounts for most of the repair in meristematic plant mitochondria and that both error-prone and accurate subtypes of DSBR lead to the observed patterns of genome evolution . One wayArabidopsis thaliana mitochondrial genome contains two pairs of very large repeats (4.2 and 6.6 kb) that commonly undergo recombination [msh1 and reca3 [One of the hallmarks of DSBR in plant mitochondria is the effect on the non-tandem repeats that exist in virtually all plant mitochondria . The Arabination ,21,22, pbination ,22,23,24nd reca3 ,26,27. TrecA homologs have been shown to be deficient in DSBR [MSH1, are transcriptionally upregulated, providing a possible explanation for the increased DSBR. We also provide additional evidence to support the hypothesis that mitochondrial DNA maintenance is abandoned in non-meristematic tissue [Numerous proteins known to be involved in the processing of plant mitochondrial DSBs have been characterized. Plants lacking the activity of mitochondrially targeted  in DSBR ,28. In a in DSBR ,15. The  in DSBR ,30, alth in DSBR . In thisc tissue , callingArabidopsis thaliana UNG T-DNA insertion strain used in this experiment, GK-440E07 (ABRC seed stock CS308282), show no uracil glycosylase activity [Arabidopsis thaliana lines and targeted the mitochondria by an amino-terminal fusion of the 62 amino acid mitochondrial targeting peptide (MTP) from the alternative oxidase 1A protein. Fluorescence microscopy of Arabidopsis thaliana expressing an MTP-A3G-GFP fusion shows that the MTP-A3G construct is expressed and targeted the mitochondria , a wild-type Arabidopsis plant expressing the MTP-A3G construct (Col-0 MTP-A3G), and a UNG plant expressing the MTP-A3G construct (UNG MTP-A3G) using an Illumina Hi-Seq4000 system. Mitochondrial sequence reads from these plants were aligned to the Columbia-0 reference genome  using BWA-MEM , and sinA. thaliana mitochondrial genome have been duplicated in the nucleus, forming regions called NuMTs, an abbreviation of Nuclear Mitochondrial DNA [There were no SNPs that reached fixation  in any plant. Mitochondrial genomes are not diploid; each cell can have many copies of the mitochondrial genome. Therefore, it is possible that an individual plant could accumulate low-frequency mutations in some of the mitochondrial genomes in the cell. VarDict was used to detect heteroplasmic SNPs at allele frequencies as low as 0.01. VarDict’s sensitivity in calling low-frequency SNPs scales with depth of coverage and quality of the sample, so it is not possible to directly compare heteroplasmic mutation rates in samples with different depths of coverage. However, because the activity of the UNG protein is specific to uracil, the absence of the UNG protein should not have any effect on mutation rates other than G-C to A-T transitions. Comparing the numbers of G-C to A-T transitions to all other substitutions should reveal if the rate of mutations that can be repaired by UNG is elevated compared to the background rate. If the UNG MTP-A3G line is accumulating G-C to A-T transitions at a faster rate than the Col-0 MTP-A3G line, we would expect to see an increased ratio of G-C to A-T transitions compared to other mutation types. Complicating the analysis, significant portions of the rial DNA ,39,40. MUNG knockout across multiple generations, we performed a mutation accumulation study [UNG homozygous plants derived from one hemizygous parent. These 23 plants were designated as generation 1 UNG and were allowed to self-cross. The next generation was derived by single-seed descents from each line, and this was repeated until generation 10 UNG plants were obtained. Leaf tissue and progeny seeds from each line were kept at each generation.To determine the effects of the on study . We chosUNG mutation accumulation lines and a wild-type Col-0 were sequenced and analyzed with VarDict as described above. Similar to the MTP-A3G plants, there were no SNPs in any of our UNG mutation accumulation lines that had reached fixation . In contrast, there was no relative increase in the ratios of GC-AT/total SNPs between the UNG lines and Col-0 . RECA3, SSB, and WHY2 showed no significant differential expression compared to wild-type (unpaired T-test p > 0.05).Because the  in DSBR ,44,45,46cox2 and rrn18 were used to standardize relative amplification between lines. We and others [UNG lines and Col-0 , supplemented by base excision repair , then ind others ,46 have d others  in both nd Col-0 b. In matUNG, MTP-A3G, and UNG MTP-A3G plants that were sequenced above were planted. Leaves were collected from each plant when it was 17 days old (young leaf) and again when it was 36 days old (mature leaf). Both the young and mature leaves of each plant were sequenced and analyzed as described above. Only 1 heteroplasmic SNP could be traced from a parent plant to both progeny, and 7 heteroplasmic SNPs could be traced from a parent plant to one progeny , leaving behind an abasic site . An AP  . Most orUNG lines. No fixed mutations of any kind were found in UNG lines, even after 11 generations of self-crossing. Low-frequency heteroplasmic SNPs were found in both wild-type and UNG lines, but UNG lines showed no difference in the ratio of G-C to A-T transitions to other mutation types when compared to wild-type. When the rate of cytosine deamination was increased with the expression of the APOBEC3G deaminase, there was an increase in G-C to A-T transitions but only in mature leaves. This is consistent with the idea of abandonment and is evidence that, in mitochondrial genomes that have not been abandoned, there is an efficient and accurate system of nonspecific repair.To determine the outcomes of genomic uracil in the absence of a functional UNG protein, we sequenced the genomes of several Arabidopsis thaliana and into the present, wild growing plants are exposed to a range of growth conditions and stresses that experimental plants in a greenhouse avoid. The rate of spontaneous cytosine deamination increases with increasing temperature [Clearly, plant mitochondria can repair uracil in DNA sufficiently to prevent mutation accumulation in the absence of the UNG protein. Why then has the BER pathway been conserved in plant mitochondria while NER and MMR have apparently been lost? DSBR may be able to protect the genome efficiently from mutations being inherited by the next generation see . There mperature ,59, so DHere, we have provided evidence that, in the absence of a dedicated BER pathway, plants growing in greenhouse growth chamber conditions do not accumulate mitochondrial SNPs at an increased rate. Instead, DNA damage is accurately repaired by double-strand break repair, which also causes an increase in ectopic recombination at identical non-tandem repeats. It has recently been shown that mice lacking a different mitochondrial BER protein, oxoguanine glycosylase, also do not accumulate mitochondrial SNPs . Here, wArabidopsis thaliana Columbia-0 (Col-0) seeds were obtained from Lehle Seeds . UNG (AT3G18630) T-DNA insertion hemizygous lines were obtained from the Arabidopsis Biological Resource Center, line number CS308282. Hemizygous T-DNA lines were self-crossed to obtain homozygous lines . All plants were grown in walk-in growth chambers under a 16:8 light:dark schedule at 22 °C. Plants grown on agar were surface sterilized and grown on 1× Murashige and Skoog Basal Medium (MSA) with Gamborg’s vitamins  with 5 μg/mL Nystatin Dihydrate to prevent fungal contamination.Arabidopsis thaliana-preferred codons and including the 62 amino acid mitochondrial targeting peptide (MTP) from alternative oxidase on the N-terminus of the translated protein. The MTP-A3G construct was cloned into the vector pUB-DEST (NCBI:taxid1298537) driven by the ubiquitin (UBQ10) promoter and transformed into wild-type and UNG Arabidopsis thaliana plants by the Agrobacterium floral dip method [Arabidopsis thaliana plants were again transformed by the Agrobacterium floral dip method, and mitochondrial fluorescence was confirmed with confocal fluorescence microscopy.The APOBEC3G gene  was syntp method . ArabidoUNG generation ten [UBQ10 and GAPDH as housekeeping gene controls. Reactions were performed in a Bio-Rad CFX96 thermocycler using 96-well plates and a reaction volume of 20 µL/well. SYBRGreen mastermix  was used in all reactions. Three biological replicates from different UNG MA lines and three technical replicates were used for each amplification. Primers are listed in RNA was extracted from young leaves of plants grown in parallel on MSA during tion ten . ReverseUNG plants grown in parallel using the CTAB DNA extraction method [cox2 and rrn18 genes were used as standards for analysis. Reactions were performed in a Bio-Rad CFX96 thermocycler using 96-well plates with a reaction volume of 20µL/well. SYBRGreen mastermix (Bio-Rad) was used in all reactions. Three biological and three technical replicates were used for each reaction. The thermocycling program for all repeat recombination qPCR was a ten-minute denaturing step at 95° followed by 45 cycles of 10 s at 95°, 15 s at 60°, and a primer-specific amount of time at 72°  magnetic beads method of Rowan et al. [ncbi.nlm.nih.gov under BioProject number PRJNA492503.DNA extraction from frozen mature leaves of Columbia-0, generation 10 and n et al. ,68. Genon et al.  and modin et al. . FollowiArabidopsis thaliana nuclear chromosomes and chloroplast genome sequences [Reads were aligned using BWA-MEM v0.7.12-r1039 . The refequences . A largeequences . To prevequences , bam filequences .Organellar variants were called using VarDict . To miniDNA damage during library preparation was measured by individually analyzing the paired ends of Illumina paired-end sequencing and by looking for imbalances in mutations between the paired ends . Mapped Nuclear variants were called using Samtools mpileup (v. 1.3.1) and Bcftools call (v. 1.2) and were filtered for SNPgap of 3, Indelgap of 10, RPB > 0.1 and QUAL > 15, at least 3 high quality ALT reads (DP4(2) + DP4(3) ≥ 3), at least one high quality ALT read per strand (DP4(2) ≥ 1 and DP4(3) ≥ 1), and a high-quality ALT allele frequency ≥ 0.3. Chromosome 2 was excluded from this analysis to avoid false positives resulting from the presence of the large NuMT that has been duplicated and repeated there.


The X-ray diffraction (XRD) confirms PbS nanocrystal structure and the atomic force microscopy (AFM) displays the crystalline phase of uniform size and distribution of PbS, indicating compact surface nanoparticles. The electrocatalytic activity by lead sulfide, using N-di-isopropyl-N-octyldithiocarbamato (OCT) without hexadecylamine (HDA) capping (OCT-PbS) was very low in HI-30 electrolyte, due to its overpotential, while lead sulfide with OCT and HDA-capped (OCT-PbS/HDA) sensitizer exhibited significant electrocatalytic activity with moderate current peaks due to a considerable amount of reversibility. The OCT-PbS sensitizer exhibited a strong resistance interaction with the electrolyte, indicating very poor catalytic activity compared to the OCT-PbS/HDA sensitizer. The values of the open-circuit voltage (OCV) were ~0.52 V, with a fill factor of 0.33 for OCT-PbS/HDA. The better conversion efficiency displayed by OCT-PbS/HDA is due to its nanoporous nature which improves the device performance and stability. Inorganic nanocrystal solar cells have been tagged as the next generation of synthesizers that have the potential to break new ground in photovoltaic cells. This synthetic route offers a safe, easy and cost-effective method of achieving the desired material. The present work investigates the synthesis of inorganic PbS sensitizers through a molecular precursor route and their impact on improving the conversion efficiency in photovoltaic cells. PbS photosensitizers were deposited on TiO The amount of sunlight energy released on a daily basis is more than sufficient if the absorbed photons are completely utilized [Research into clean energy has been one of the main concerns of material scientists. The release of environmental pollutants, such as COutilized . Apart futilized ,2. This utilized ,4,5,6,7.Inorganic nanocrystal solar cells have been tagged as the next generation of sensitizers that have the potential to break new ground, using semiconductors such as PbS . The adoThe XRD patterns of OCT-PbS/HDA and OCT-PbS photosensitizers are illustrated in The HRTEM images of OCT-PbS and OCT-PbS/HDA as seen in 2 photoanode, it is compact and smooth. These results can be linked to the densification and reorganization of the crystallites of the OCT-PbS/HDA film. The photosensitizers exhibited particle size of 0.654 μm for OCT-PbS/HDA and 1.69 μm for OCT-PbS, depicting a regular crystal growth rate. The height clusters of both samples depicted an insignificant change around 0.328 and 1.35 μm, which connotes smaller nanocrystals [The surface roughness of the OCT-PbS and OCT-PbS/HDA photosensitizers is indicated by the AFM images see . The AFMIn order to evaluate electrocatalytic abilities and the reaction kinetics of PbS/HDA and PbS photosensitizers, the cyclic voltammetry (CV) measurements were adopted with three-electrode systems . The obs1). At low frequencies, the impedance related to the charge transport at the TiO2/PbS/electrolyte interface is R2. This study focused only on the R2 to compare the effect of the HDA capping agent on the charge transfer and transport at the TiO2/PbS/electrolyte interfaces. The impedance at low frequencies can be pinpointed using R2. When the R2 is lower, the charge transfer is faster at the photosensitzer/electrolyte interface. OCT-PbS/HDA indicated a very poor catalytic activity, which can be linked to the injection of HDA. On the other hand, the OCT-PbS sensitizer displayed lower charge transfer at the photosensitzer/electrolyte interface. This can be linked to the particle size of OCT-PbS sensitizer, which promotes the charge transport speed of the solar cell. This further affirmed the CV and AFM result [2 is also considered as a resistance of charge recombination at the interfaces of TiO2/QDs/electrolytes. Decreases in R2 can boost the charge recombination and shorten electron lifetime, which causes the performance of the solar cell to deteriorate.M result ,41. HowerΤ) can be estimated with Equation (1) using the Bode plot.According to the EIS diagrams, shown in rΤ value of 57 ms. This indicates an increase in electron lifetime and a backreaction reduction of the electron with the injected HI-30 electrolyte. The suppressing of charge recombination implies the longer lifetime of the charge carrier. This leads to better electron collection at the FTO substrate. Based on this study, we can conclude that OCT-PbS displays superior ability compared to OCT-PbS/HDA. Further, the complete coverage of photosensitizers on a photoanode surface is directly linked to their ability to inhibit the charge recombination back to the electrolyte redox couple [The OCT-PbS exhibited a x couple .J−V) characteristics of the QDSSCs of OCT-PbS/HDA and OCT-PbS photosensitizers under illumination. A reasonably high short-circuit current density (SCJ) of 11 mA/cm2 was observed in the OCT-PbS/HDA QDSSCs, compared to that of the OCT-PbS cells. The nanoporous nature of OCT-PbS/HDA may be related to the high value of SCJ, due to its fabrication by the single-source precursor technique. The values of the open-circuit voltage (OCV) (~0.52 V) and efficiency (1.89) for OCT-PbS/HDA proved that the addition of HDA resulted in improved solar cell parameters. The TiO2 nanoparticle photoanode, with broad size variation and far-field and near-field effect, led to enhancement of SCJ in OCT-PbS/HDA photosensitizer. This also exhibited high absorption of light in the whole visible spectrum. Secondly, recombination was reduced when PbS nanoparticles functioned as electron scavengers [2 conduction band could be linked as a factor for higher Voc value of OCT-PbS/HDA (0.52 V) compared to that of OCT-PbS (0.48 V), which elevated the Fermi level [avengers ,45. The mi level . The FF mi level . The higmi level .2, platinum FTO, HI-30 electrolyte iodide, masks, gaskets, chenodeoxycholic acid (CDC) and a hot seal were purchased from Solaronix Company . Additionally, water, oleic acid (OA), methanol, hexadecylamine (HDA), bis(N-diisopropyl-N-octyldithiocarbamato) (OCT) Pb(II) complexes were obtained from Merck .All materials were purchased from commercial sources and used without further purification. The complete test kits containing fluorine-doped tin oxide (FTO) as glass substrate of TiObis(N-diisopropyl-N-octyldithiocarbamato) Pb(II) complex was dissolved in 4 mL oleic acid (OA) and injected into 3 g of hot hexadecylamine (HDA) at 360 °C. An initial temperature of 20–30 °C was attained for the mixture. The reaction was stabilized at 360 °C, and the process lasted for 1 h. The process was allowed to drop to 70 °C, signifying the completion of the process, and about 50 mL of methanol was used to remove excess OA and HDA. Centrifugation was used to separate the flocculent precipitate, which was redispersed with toluene. Low air pressure was used to remove solvent, giving rise to metal sulfides of OCT-PbS/HDA nanoparticles. Synthesis of OCT-PbS nanocrystals was obtained according to the study by a co-worker [−1 temperature gradient between 30–900 °C. A purge gas of flowing nitrogen at a rate of 20 mL min−1 was used. At temperatures between 360 and 900 °C, the complex end-product was converted into the residue of OCT-PbS nanoparticles from the TGA. Nanoparticles were fabricated according to methods in the literature : 0.20 g o-worker , using , with 6 × 6 mm2 active areas of TiO2 screen-coated. Dye loading for sensitization was done using 10 mL of warm water with OCT-PbS/HDA and OCT-PbS. Co-adsorbents (co-adsorbent/dye), using chenodeoxycholic acid (CDC), were added. The mediating solution was a commercial HI-30 electrolyte solution , with content of iodide species at 0.05 M. The TiO2 thin film was soaked in a solution of photosensitizers for 24 h. The two substrates, one coated with TiO2 loaded with photosensitizers and the other with platinum, were held together using polyethylene and a soldering iron. A syringe was used to inject the HI-30 electrolyte (iodide).Quantum dot solar cells (QDSC) were prepared with 2 × 2 cm2 was used as the anode, while HI-30 iodide electrode was utilized as a reference electrode. Cyclic voltammetry (CV) was performed at scan rates between 0.05 to 0.35 V s−1 with an increment of 0.05 V s−1. Electrochemical impedance spectroscopy (EIS) was carried out in the frequency range of 100 kHz to 100 mHz. Current–voltage (J−V) parameters were collected through a Keithley 2401 source meter and a Thorax light power meter (RS Components (SA), Johannesburg, South Africa). A Lumixo AM1.5 light simulator was employed, and the lamp was fixed at 50 cm high to avoid illumination outside of the working area. To avoid cell degradation, temperature was kept below 60 °C, and the light power density was kept at 100 mW/cm−2 (AM1.5). A PerkinElmer Lambda 25 UV−Vis spectrophotometer was employed to carry out observations of optical absorption properties at room temperature . An X-ray diffractometer  was employed to evaluate the structural pattern of the samples; the diffraction structure results were recorded between 10 to 90° at intervals of 0.05°. A JEOL JEM 2100 high-resolution transmission electron microscope  (HRTEM) operating at 200 KV with selected area electron diffraction (SAED) patterns was used. The surface roughness of the OCT-PbS/HDA and OCT-PbS FTO substrates were identified through the use of atomic force microscopy  in contact mode and a scan rate of 0.8 Hz. Electrochemical studies were evaluated by Metrohm 85,695 Autolab with Nova 1.10 software  Ltd.). A platinum electrode was adopted as a counter electrode. TiO2 by direct deposition revealed the structure, morphologies and electrocatalytic activity of a typical PbS nanocrystalline structure and displayed uniform size-distribution. The SAED of OCT-PbS and OCT-PbS/HDA confirmed the polycrystalline nature of both materials. The AFM results of OCT-PbS/HDA suggested that the injection of the HDA capping agent improved the surface roughness of the materials with TiO2 photoanode. Electrocatalytic activity of OCT-PbS was enhanced in HI-30 electrolyte compared to the OCT-PbS/HDA sensitizer and exhibited significant electrocatalytic activity. The OCT-PbS/HDA sensitizer exhibited a strong resistance interaction with the electrolyte, indicating poor catalytic activity, compared to the OCT-PbS sensitizer. The better conversion efficiency displayed by OCT-PbS/HDA proved its superiority as a good sensitizer, and this is strongly linked to its nanoporous nature and electrocatalytic activity. The addition of an HDA capping agent has made a great improvement to the conversion efficiency of OCT-PbS/HDA. In summary, the fabrication of inorganic PbS sensitizers through the molecular precursor route and their application in photovoltaic cells were investigated in this study. PbS photosensitizers deposited on TiO


Artemisia annua. Such therapeutics are very effective but show heavy side effects like drug resistance. In this study, “green” silver nanoparticles (AgNPs) have been prepared from two Artemisia species (A. abrotanum and A. arborescens), traditionally used in folk medicine as a remedy for different conditions, and their potential antimalarial efficacy have been assessed. AgNPs have been characterized by UV-Vis, dynamic light scattering and zeta potential, FTIR, XRD, TEM and EDX. The structural characterization has demonstrated the spheroidal shape of nanoparticles and dimensions under 50 nm, useful for biomedical studies. Zeta potential analysis have shown the stability and dispersion of green AgNPs in aqueous medium without aggregation. AgNPs hemocompatibility and antimalarial activity have been studied in Plasmodium falciparum cultures in in vitro experiments. The antiplasmodial effect has been assessed using increasing doses of AgNPs (0.6 to 7.5 μg/mL) on parasitized red blood cells (pRBCs). Obtained data showed that the hemocompatibility of AgNPs is related to their synthetic route and depends on the administered dose. A. abrotanum-AgNPs (1) have shown the lowest percentage of hemolytic activity on pRBCs, underlining their hemocompatibility. These results are in accordance with the lower levels of parasitemia observed after A. abrotanum-AgNPs (1) treatment respect to A. arborescens-AgNPs (2), and AgNPs (3) derived from a classical chemical synthesis. Moreover, after 24 and 48 hours of A. abrotanum-AgNPs (1) treatment, the parasite growth was locked in the ring stage, evidencing the effect of these nanoparticles to hinder the maturation of P. falciparum. The anti-malarial activity of A. abrotanum-AgNPs (1) on pRBCs was demonstrated to be higher than that of A. arborescens-AgNPs (2).Malaria represents one of the most common infectious diseases which becoming an impellent public health problem worldwide. Antimalarial classical medications include quinine-based drugs, like chloroquine, and artesunate, a derivative of artemisinin, a molecule found in the plant  Plasmodium into the host  _ 100%.The pRBCs cultures previously obtained were provided in order to perform the hemolysis assay. Fresh RBCs were prepared to maintain the pRBCs cultures at 1% haematocrit. Fresh human heparinized whole blood was obtained from healthy volunteer donors. RBCs were purified from blood by centrifugation at 200 g for 5 min to remove plasma and leukocytes. RBCs were then washed three times in sterile complete growth medium as previously described –42. ParaP. falciparum were treated for 24 and 48 hours in the presence of the indicated concentrations of all the tested silver nanoparticles: A. abrotanum-AgNPs (1), A. arborescens-AgNPs (2), AgNPs (3). All compounds were freshly prepared before any experiment and used immediately.1 mg/mL of silver nanoparticles was serially diluted prior to addition to malaria cultures in PBS 1X and 5 mM glucose. Untreated cultures were run in parallel with the same final concentration. Cultures at the ring stage and fresh isolates of The morphology and the total parasitemia assays were performed using a standard method –42. Para50 values using a nonlinear regression function of the R software.To calculate the half maximal inhibitory concentrations of the different silver nanoparticles, ICEstimator software version 1.2 was used. The program estimates ICData analyses were performed using Prism GraphPad software. Statistics for experiments were performed using a t-test. In all cases * was used for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Values were expressed as the mean ± SD. All experiments were performed at least in triplicate.A. annua (different extracts-to-silver ratio) as summarised in  useful for biological applications [“Green” AgNPs have been prepared from leaf extracts using a modified method  for analrised in , which sications . Howeverications . Cell upications .Artemisia leaf extract was carried out by a series of spectroscopic analyses. The UV-Vis spectra show the AgNPs surface plasmon resonance effect which reflects the method of synthesis, in turn affecting nanoparticle shape and size. Data results indicate that the peak observed in the range 400–450 nm was increased in A. abrotanum-AgNPs (1) and A. arborescens-AgNPs (2) respect to “classical” AgNPs (3), suggesting a spheroidal shape for these nanoparticles with a size below 50 nm [A. abrotanum-AgNPs (1), and 30 nm for A. arborescens-AgNPs (2), respectively (A. abrotanum-AgNPs (1) and A. arborescens-AgNPs (2) have shown a good zeta potential value ..Artemisiectively . All “grectively . In ordeal value  underlinal value  Figs. Coal value  evidenciThe evaluation of the structural features of the nanoparticle surface plays a significant role in the understanding of the possible effects of AgNPs on the cell membrane for biological applications.Artemisia leaf extracts. The very broad band going from 3100 to 3600 cm-1 is encompassing the OH and NH stretching frequencies, the signals around 2900 cm-1 are due to the CH stretching mode of hydrocarbon moieties, while those around 1620 (ν C = O) are most probably due to the carbonyl group of amidic compounds (i.e. proteins and enzymes). A series of bands can be attributed to polyphenols: 2920 cm-1 (stretching of the C-H bond adjacent to a quinone moiety), 1440 cm-1 (stretching of the C = C bond adjacent to the quinone system) and 1250 cm-1 (stretching of the C = C bonds of the various coupled aromatic systems). Moreover, the band in the range 1400–1380 cm-1 may be attributed to silver nanoparticles in accordance with the literature [-1 is due to water molecules, while the peaks in the regions 1600–1500 and 1400–1300 cm-1 can be attributed to the overlapping of the COO- asymetrical and symmetrical stretching vibrations, respectively, of citrate and ascorbate anions present on the nanoparticle surface [FT-IR spectra  were recterature . As for A comparison between the FTIR of the dry plant extracts and the relative AgNPs has been undertaken to add more information about the capping. The two sets of spectra are similar but not identical, showing that most but not all the biomolecules present in the plant extract have contributed to the formation of the capping .et al. [The XRD patterns showed a progression of broadened line profiles which was matched with the presence of Ag metal phase with space group Fm-3m and a = 4.0853 (4 Ag atoms in the unit cell). In et al.  is similThe hemolysis assay has commonly been performed at body and fever temperature  at concentrations ranging from 0.6 to 7.5 μg/mL up to 48 hours of incubation .p < 0.01). In particular, A. arborescens-AgNPs (2) have shown an evident effect on pRBCs at 5 and 7.5 μg/mL dosage (p < 0.01 and 7 μg/mL p < 0.001 respectively for both temperature conditions) compared to A. abrotanum-AgNPs (1), probably due to the different capping and size  and A. arborescens-AgNPs (2) have demonstrated significant in vitro activity against P. falciparum in pRBCs, showing dose-dependent hemolytic effect (A. arborescens-AgNPs (2) showed antiplasmodial activity by blocking the parasite maturation stage from trophozoite to rings (A. abrotanum-AgNPs (1) have demonstrated their antiplasmodial activity achieving the parasite death compared to the control after 24 and 48 hours of treatment (A. abrotanum-AgNPs (1) showed the best antiplasmodial activity considering the IC50,90,99 as reported in The next experiments have been carried out using the non hemolytic concentration of nanoparticles as previously described. In particular, pRBCs were treated with increasing doses of all nanoparticles (0.6 to 7.5 μg/mL) at both 24 and 48 hours in order to evaluate the effect of parasite growth inhibition. Palo Alto (PA) has been used as a representative strain as no significant differences have been observed among the other strains tested . c effect  connectec effect  comparedc effect . Consideto rings . On the reatment . MoreoveA. abrotanum-AgNPs (1) have demonstrated a lower aggregation in aqueous medium and better shape and dimension compared to the other nanoparticles taken into consideration. In order to investigate each AgNPs type effects on PA cultures, their chemical structure has been studied and correlated with their activity and biocompatibility. Indeed, to study the effect of “green” AgNPs on RBCs biocompatibility, several in vitro experiments have been performed on human pRBCs infected by P. falciparum. After the nanoparticles characterization, their hemolytic properties in contact with the human erythrocytes were studied. The increase in hemolytic activity observed is probably due to the higher temperature used for fever simulation, where the RBCs could be sensitive to the temperature of 41°C. On the other hand, based on the large hemolysis dependence on fever temperature, non-hemolytic concentrations were assessed below 5 μg/mL. It is noteworthy that after 24 hours of incubation there was no further increase of the hemolytic activity. The different hemolysis effect could be assigned to the difference in the size of nanoparticles and/or the different biomolecules present in the capping, which cannot be excluded. In fact, it has been reported that the variety of shape, size and chemistry of nanoparticles produce different effects on the biological environment [It is known that a critical step in biomedical applications of nanoparticles is to study their physical-chemical properties in order to correlate their biological activity to specific parameters such as composition, size, shape and capping. In this study, considering the same concentration of nanoparticles used for the analysis, the difference observed during the evaluation of each nanoparticle activity may depend on these parameters. In this context, ironment .P. falciparum pRBCs. The striking morphological alteration observed in fixed blood smears of PA strain treated with A. abrotanum-AgNPs (1) and A. arborescens-AgNPs (2), is again most probably linked to the small size and the different nature of synthesis using Artemisia extracts. Despite the smaller size of A. arborescens-AgNPs (2) respect to the other nanoparticles, which should confer them higher efficacy, their lower activity against the parasite may depend on the different capping from the A. arborescens plant extract used.However, to further evaluate the effects on parasite maturation and death, a morphology investigation was performed on different strains of P. falciparum parasite. In general, all the results gathered in this study underline the higher antiplasmodial efficacy of AgNPs from A. abrotanum extract respect to the A. arborescens ones.Anyway, this method of synthesis has indeed allowed to create small size nanoparticles useful for biomedical applications connected to an effective action against Artemisia sp. derived silver nanoparticles potential antimalarial efficacy in parasitized human red blood cell and to understand their efficacy against P. falciparum as a new nanotool against malaria. The obtained results have demonstrated that plant extracts derived from A. abrotanum and A. arborescens can be used for the facile biosynthesis of silver nanoparticles and to create a bio-capping on the AgNPs useful to modulate their activity against P. falciparum cultures in in vitro experiments. Results evidenced a high anti-malarial activity for A. abrotanum-AgNPs (1) in compared to A. arborescens-AgNPs (2). Their hemolytic effect is dose-dependent, and is more pronounced for the smallest AgNPs, i.e. those derived from A. arborescens, as expected. The high anti-malarial activity for A. abrotanum-AgNPs (1) is connected to the small size of nanoparticles and their different effect on the parasite cycle. In fact, results underlined that A. abrotanum-AgNPs (1) were been able to hinder the stage of the maturation of the parasite locking it in the ring stage after treatment. The mechanism behind this behaviour is still unknown. Studies reporting the biological bases of antiprotozoal action of silver nanoparticles are very rare [Artemisia-derived AgNPs in in vitro experiments against P. falciparum is promising and deserves further research.The present work was performed as a pilot study in order to evaluate ery rare , so the S1 FigA. abrotanum-AgNPs (1), A. arborescens-AgNPs (2) and AgNPs (3). Cu peaks presented in the graphs are due to the grid used.Evaluation of nanoparticles silver signal using EDX profile of (TIF)Click here for additional data file.S2 FigA. abrotanum-AgNPs (1) stability and dispersion in aqueous medium.Evaluation of (TIF)Click here for additional data file.S3 FigA. arborescens-AgNPs (2) stability and dispersion in aqueous medium.Evaluation of (TIF)Click here for additional data file.S4 FigEvaluation of AgNPs (3) stability and dispersion in aqueous medium.(TIF)Click here for additional data file.


Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via  In recent decades, public misunderstanding about the use of antibiotics, the lack of strict distribution standards between clinics, and the addition of antibiotics to animal feed have led to an abuse of antibiotics, resulting in a rapid increase in antibiotic bacterial resistance. Researchers are, therefore, committed to developing new forms of antibiotics to replace the current pharmaceutical options. Existing data show that antibacterial peptides are the most promising candidates. Antimicrobial peptides (AMP) possessing antibiotic properties are widely distributed in nature, including in animals , insectsCecropin is a non-specific AMP mostly derived from insects, arthropods, and invertebrates . It is cSome studies reported on the use of microbes as hosts for the production of AMPs ,14,15,16In our previous study, the production of cecB2 peptide was performed with the construction of a fusion protein with CBD-INT . We founEscherichia coli ER2566 was selected as the host due to its lack of outer membrane proteases [E. coli ER2566 competent cells via heat shock.To construct the fusion proteins, a T vector carrying the cecB2 gene was used as a template and various primers were designed  to ampliTo test the recombinant constructs, the recombinant strains with various pTWIN1-CBD-INT-OCV-cecB2 were subjected to protein expression following the procedure outlined in For the INT cleavage test, the harvest supernatant was subjected to filtration to remove the proteins with a molecular weight of less than 10 kDa, as depicted in In this study, an attempt was made to find out whether INT could be treated as a catalyst for the self-cleavage reaction; in this way, the kinetics of INT cleavage reactions were tested. From the time courses of the cleavage reactions for various OCVs shown in In order to determine the best conditions, the fusion protein CBD-INT-SRA-cecB2 was selected and subjected to various cleavage conditions . As per NEB’s instructions, a cleavage buffer  was used. It also indicated that the most suitable pH for cleavage at the C-terminus was pH 6.0–7.5; a pH higher than 8.0 or lower than 5.5 would have suppressed the cleavage reaction. In the literature, the optimal condition for cleavage was reported to be pH 7.0 . The mixTo test the effect of temperature on INT cleavage, various temperatures were applied . The cleavage reaction was set at pH 6.0 for 1 d. The results are shown in E. coli cultivation, the obtained supernatant with the fusion protein CBD-INT-SRA-cecB2 was subjected to a CBD affinity column, followed by washing, elution, and stripping procedures. The results of the SDS-PAGE are shown in To test the in-column purification of the fusion proteins after the E. coli strains. The serial dilution number of the purified cecB2 (58.7 mg/L) was detected in triplicate and is shown in E. coli JM109 > E. coli BL21 > E. coli DH5α. Although the antibacterial activity might be a little different for the strains belonging to the same species, in this type of assay, minimum inhibition concentration (MIC) values differing by a factor of two are assumed to be identical. The calculated MIC values were in the range of 1.8–3.7 (mg/L), equivalent to 0.45–0.90 μM. Wang et al. reported that the synthesized cecropin B showed an MIC of 0.78–1.56 μM to various E. coli strains [In previous reports, the cecropin family was reported to exhibit various antimicrobial effects against Gram-negative strains, Gram-positive strains  and even strains , which iIn this study, it was found that the fusion protein with an OCV of SRA inserted into the INT C-terminus was shown to promote the self-cleavage reaction of the intein. According to the scheme, the cleavage reaction may be affected by two factors—the structural stability of the cleavage sequence and the chemical environment near the peptide bond. A stable conformation of the cleavage sequence will help the cyclization of Asn via sidechain NH2 attack A. In addTo explore the situation in the cleavage reaction, all-atom MD simulations of these OCVs were carried out. The structural stability of the cleavage sequence was evaluated via the root-mean-square fluctuations (RMSF) for Cα atoms from the 10-ns simulation trajectory files . The RMSFrom the MD simulation snapshots , it was E. coli strains. In addition, some advantages for the construction of INT with SRA was highlighted via the MD simulation. In conclusion, the presence of the CBD-INT-SRA domain might not only accelerate the INT self-cleavage reaction, but also make product purification easier via the in-column cleavage reaction. The process developed in this study may provide a reference for other similar AMP production and for future large-scale applications.In this study, inteins with various OCVs were constructed and their effect on INT self-cleavage was tested for cecB2 production. When comparing various OCVs, SRA gave the best result for facilitating the INT cleavage reaction. Assuming INT as a biocatalyst, a first-order reaction rate equation was found to be a good tool for predicting the kinetics of the self-cleavage reaction. The in-column purification could be properly applied to the preparation of cecB2, expressing antimicrobial activity against various E. coli DH5α and E. coli ER2566 were used as host cells for gene transfer and recombinant protein expression, respectively. The plasmid pTWIN1 containing the CBD-INT gene sequence and the mouse monoclonal anti-CBD were obtained from NEB . The plasmid containing the prepro-cecropinB2 gene sequence was provided by Professor Guangwu Du at National Chung Hsing University. The E. coli host strains and vectors used in this study are listed in The prepro-cecB2 template was applied in PCR to amplify the cecB2 gene carrying various OCV sequences. The primers are listed in E. coli ER2566 hosts carrying various fusion protein genes were activated from preservation in a petri dish with 10 mL of LB culture agar medium plus 10 μL (50 mg/mL) ampicillin. Two colonies from the dish were picked to inoculate 5 mL LB medium plus 5 μL (50 mg/mL) ampicillin, followed by overnight cultivation at 37 °C and a shaking speed of 200 rpm. One milliliter of culture broth was used to inoculate 100 mL of LB culture medium plus 100 μL (50 mg/mL) ampicillin. Cultivation was conducted at 37 °C and 200 rpm until cell optical density (OD600) reached 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a concentration of 0.4 mM, and then cultivation continued at 15 °C and 200 rpm for another 16 h. The harvested cells were subjected to centrifugation and disruption for further protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot testing for CBD.The harvested cell pellet was resuspended in Tris HCl buffer  and then lysed by sonication, using an ultrasonic processor at 20 W for 10 cycles , to obtain the crude cell extract. The supernatant was collected and applied to a chitin column, followed by rinsing with the washing buffer . The cleavage buffer  was then loaded and the sample kept at 25°C overnight to obtain cecB2. The column was cleaned by loading stripping buffer (0.3 M NaOH) for 30 min to remove the bound CBD-INT segment. The eluted cecB2 was stored at 4 °C until further use. For the INT cleavage test, the crude cell extract was concentrated via a filter . The upper retentate was collected and mixed with the cleavage buffer under various cleavage conditions.Protein analysis was carried out using 15% SDS-PAGE and Coomassie Blue staining . The Tri600 = 0.7) was added to 6 mL of sterilized LB broth containing 0.8% agar, followed by pouring it into a petri dish (9.0 cm) to form an agar with a 1 mm depth. To prepare the sample, the purified AMP sample was diluted following the serial two-fold dilutions. In the test, a volume of 20 μL of the diluted sample was added to the surface of the agar and the petri dish was incubated at 37 °C for 12 h. Antimicrobial activity was observed by the clear zone around the testing spot. The minimal serial dilution number was set where no clear zone was observed. Each assay was performed in triplicate and the average serial dilution number was calculated. The minimum inhibition concentration (MIC) was defined as the least-diluted concentration processing antimicrobial effect [Antimicrobial activity was modified from the agar diffusion test . In briel effect .Bacillus circulans WL-12 (PDB ID: 1ED7), the crystal structure of DnaB INT from Synechocystis sp. PCC 6803 (PDB ID: 1MI8), and the solution structure of the GK cecropin-like peptide from Aedes aegypti (PDB ID: 2MMM). The initial template of cecB2 was modeled via the Phyre2 server [Prior to the MD simulation, the structural model of CBD-INT-OCVs-cecB2 fusion protein had to be built . Based o2 server . The ZDO2 server  was appl2 server , and the2 server  and PROC2 server  with theAfter the protein model was built, it was subjected to an MD simulation using GROMACS version 4.6.7 with OPLS-AA force field in the TIP3P explicit solvent model . The prohttp://pldserver1.biochem.queensu.ca/~rlc/work/pymol/). The solvent accessibility of the protein variants at residue level was calculated using POPS [The local motions of the OCVs  were studied using MD simulation. The setup included a force field, solvent model, and the same parameters as the aforementioned 400 ns MD simulation for GRA. Each protein variant was subjected to a 20 ns MD simulation. After a 10 ns equilibration phase, the last 10 ns trajectories were analyzed using GROMACS utilities. The root-mean-square fluctuation (RMSF) was calculated using the rmsf_states.py script in PyMOL (ing POPS . A 20% cing POPS . All string POPS .k is the kinetic rate constant in unit (d−1) and c is the intein concentration. The kinetic constant of Equation (1) with an initial condition of c = c0 (0t = 0) is expressed asA first-order kinetic model was used to describe intein cleavage kinetics, as follows:The first-order reaction kinetics is based on the reaction mechanism that only one reactant is involved in the reaction rate-limiting step, which is quite proper to describe the intein cleavage reaction as shown in To obtain the statistical results, all data were analyzed by Origin software, version 9.0. Experiments were performed in triplicate and expressed in mean ± SD; the significant difference between means was identified via analysis of variance .


The stromal-cell-derived factor-1α (SDF-1) is well-known for playing important roles in the regeneration of tissue by enhancing cell migration. However, the effect of SDF-1 in meniscal healing remains unknown. The purpose of this study is to investigate the effects of intra-articular injection of SDF-1 on meniscus healing in a rat meniscal defect model. The intra-articular SDF-1 injection was performed at meniscectomy and one week later. Macroscopic and histological assessments of the reparative meniscus were conducted at one, two and six weeks after meniscectomy in rats. In the macroscopic evaluation, the SDF-1 group showed an increase in the size of the reparative meniscus at six weeks after meniscectomy compared to the phosphate-buffered saline (PBS) injection (no-treatment) group. Histological findings showed that intra-articular injection of SDF-1 enhanced the migration of macrophages to the site of the regenerative meniscus at one and two weeks after meniscectomy. CD68- and CD163-positive cells in the SDF-1 group at one week after meniscectomy were significantly higher than in the no-treatment group. CD163-positive cells in the SDF-1 group at two weeks were significantly higher than in the no-treatment group. At one week after meniscectomy, there were cells expressing mesenchymal-stem-cell-related markers in the SDF-1 group. These results indicate the potential of regenerative healing of the meniscus by SDF-1 injection via macrophage and mesenchymal stem cell accumulation. In the present study, intra-articular administration of SDF-1 contributed to meniscal healing via macrophage, CD90-positive cell and CD105-positive cell accumulation in a rat meniscal defect model. The SDF-1–CXCR4 pathway plays an important role in the meniscal healing process. For potential clinical translation, SDF-1 injection therapy seems to be a promising approach for the biological augmentation in meniscal injury areas to enhance healing capacity. A meniscus is a crescent-shaped fibrocartilage tissue in the knee joint . It is aStromal-cell-derived factor-1 (SDF-1) is a chemokine also known as C-X-C motif chemokine ligand 12. SDF-1 is a potent chemotactic cytokine with various biological functions, such as inflammatory cell infiltration, angiogenesis and stem cell migration ,8. It haSeveral studies have investigated the potential of different strategies for enhancing meniscal healing, including the use of growth factors , stem ce+, CD163+) accumulation, the same as a wound-healing mechanism  wa wap = 0.p = 0.01)  staining, a mixture of round and spindle-shaped cells was observed in the regenerated meniscus one week after meniscectomy in both groups. Toluidine blue staining revealed no metachromasia in either group. Immunohistochemistry revealed a few CD90- and CD105-positive cells only in the reparative tissue of the SDF-1 group at one week a. At six = 0.01) c.+, CD163+ and α-smooth muscle actin (αSMA)+ cells, each cell’s number was estimated. One week after meniscectomy, CD68-positive and CD163-positive cells in the SDF-1 group were significantly higher than in the no-treatment group  (p = 0.84) (p = 0.015). For CD68-positive cells and αSMA-positive cells, no significant difference was noted between the two groups . At six weeks after meniscectomy, the percentage of CD68-positive cells and CD163-positive cells decreased to approximately 1%.To assess the effect of the SDF-1 injection on CD68 = 0.04) a,b. For  = 0.84) c. Two weThis study revealed that intra-articular injection of SDF-1 promoted the migration of MSC-related markers and CD68- and CD163-positive macrophages at one week and a CD163-positive macrophage at two weeks in a rat meniscal defect model. It resulted in an improvement of the macroscopic and histological findings of reparative meniscal tissues at six weeks by SDF-1 intra-articular injection.It was reported that vascularity at the injured site strongly influences the outcome of meniscus healing . AdditioPreviously, SDF-1 was shown to attract a myriad of cell populations, such as monocytes, lymphocytes and hematopoietic and bone marrow mesenchymal stem cells (BMSCs), to the injury site ,8. In thPreviously, many studies have reported that intra-articular injection of cells is an effective treatment strategy for regenerating an injured meniscus. Murphy et al. found that local delivery of adult MSCs to injured knee joints could stimulate the regeneration of meniscal tissue . Horie eMSCs have been identified as both CD90- and CD105-positive cells . Wang et+ subpopulation of synovial fluid mesenchymal progenitor cells has a higher chondrogenic potential compared to that of the CD90- population [The study has several limitations. First, the concentration of SDF-1 at the injured site could not be measured, and we did not evaluate the variation of the effect due to the concentration. Second, we proved that accumulation of M2 macrophages were increased by intra-articular injection of SDF-1. However, the amount of cytokines produced by M2 macrophages, such as IL-10 or TGF-β, was not measured. Third, we did not prove where these effective cells come from because a parabiosis model was not used in this study. As a previous study showed that macrophages and MSCs were migrated into the injured site from bone marrow, these cells would be suspected to come from bone marrow. Fourth, double staining of CD90- and CD105-positive cells was not examined in this study. A previous study demonstrated that CD90-positive cells play a crucial role in allowing cells to differentiate into multiple cell phenotypes . Additiopulation . Fan et pulation . CD90- apulation ; the dosThirty-four female Lewis rats (eight weeks old) were obtained from Nihon SLC . The animals were fed in cages with free access to food and water in an air-conditioned environment. All animal procedures were approved and conducted in accordance with the regulations of the Osaka City University Graduate School of Medicine Committee on Animal Research .N = 17). In the no-treatment groups (N = 17), 200 ng/50 μL of PBS  instead of SDF-1α was injected into the left knee. Thereafter, skin incisions were closed using 5–0 nylon sutures. Rats were allowed to walk freely in their cages after surgery. One week postsurgery, five rats from each group were sacrificed via CO2 inhalation. The remaining rats in each group  were reinjected with 200 ng/50 μL of SDF-1α (or PBS) into the left knee joint. They were then sacrificed via CO2 inhalation at two (N = 5) and six (N = 7) weeks after partial meniscectomy. The study protocol is shown in The animals were anesthetized via subcutaneous injection of ketamine  and xylazine  at a ratio of 10:3 and a dose of 1 mL/kg body weight, as previously described ,18. A stThe tibial plateau with medial reparative menisci was carefully separated from the femoral condyle at six weeks after meniscectomy. Macroscopic pictures of the meniscus with the tibia were taken using a stereomicroscope . Quantification of the size of the regenerated meniscus was performed using ImageJ software  to measure the ratio of the whole area of the medial meniscus, including both the regenerated region and normal region, to the whole area of the medial tibial plateau . ThereafThe reparative meniscus at one and two weeks after meniscectomy was carefully removed from the femur and tibia. The meniscus at six weeks after meniscectomy was also used for histological examination after photography for macroscopic evaluation. Regenerated meniscal tissues were fixed in 4% paraformaldehyde for 24 h and dehydrated in 10% sucrose for 4 h, 20% sucrose for 4 h and 30% sucrose overnight at 4 °C. Thereafter, tissues were frozen and stored in the dark at −80 °C after being embedded in optimal cutting temperature (OCT) compound . Specimens were cut radially at 5 µm thickness using a cryostat. Specimens from one week after meniscectomy underwent immunofluorescence staining, and those from six weeks were stained with hematoxylin and eosin, toluidine blue or safranin O and Fast Green for histological evaluation of cell morphology and meniscus regeneration. For hematoxylin and eosin staining, sections of the harvested tissues were rinsed in PBS for 5 min at room temperature , and then the slides were stained with Mayer’s hematoxylin  for 10 min, and after being rinsed in PBS, the slides were stained with eosin  for 10 min. For toluidine blue staining, sections of the harvested tissues were rinsed in PBS for 5 min at RT, and then the slides were stained with toluidine blue solution  for 15 min. For safranin O and Fast Green staining, sections of the harvested tissues were rinsed in PBS for 5 min at RT, and then the slides were stained with 0.1% Fast Green  for 5 min and stained with 0.1% safranin O . In all stains, the material was dehydrated with ethanol and sealed after permeation with xylene. Microscopic evaluation was performed using the Olympus BX53 . The regenerated meniscus at six weeks after meniscectomy was evaluated using the quantitative score based on the modified Pauli’s score . The modCD90 and CD105 staining was performed to confirm the presence of MSC for the meniscus at one week after meniscectomy, and type II collagen staining was performed to confirm the presence of type II collagen in the reparative meniscus six weeks after meniscectomy in the SDF-1 and no-treatment groups, respectively. Immunohistochemistry staining was performed with reference to the previously reported method ,45. To pThe regenerated meniscus at one, two and six weeks after meniscectomy was used for evaluation. Immunofluorescence staining was performed as previously reported . In prepFor immunofluorescent quantification, DAPI and specific markers of interest were counted from each meniscal section in three randomly selected areas under high power (400× magnification). To determine the proportion of cells stained positive by primary antigens, the following formula for the positive cell rate was used:  × 100 (%), where X = CD68, CD163 or αSMA.http://www.r-proje ct.org, The R Foundation, Vienna, Austria) software package, and significance was assumed at p < 0.05.The Mann–Whitney U test was used for all comparisons between the SDF-1 and no-treatment groups. This included the Pauli score, meniscus covering ratio and modified Pauli score at six weeks after meniscectomy as well as CD68-, CD163- or αSMA-positive cell rates at one, two and six weeks after meniscectomy. Analysis was performed with the R (version 3.5.1, patched, In the present study, intra-articular administration of SDF-1 contributed to meniscal healing via macrophage, CD90-positive cell and CD105-positive cell accumulation in a rat meniscal defect model. The SDF-1–CXCR4 pathway plays an important role in the meniscal healing process. For potential clinical translation, SDF-1 injection therapy seems to be a promising approach for biological augmentation in meniscal injury areas to enhance healing capacity.